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Using this model pathogen, we performed an in-depth analysis of the quantitative expression stability of the Th1 key transcription factor T-bet. Previously, it was shown that virus-specific Th1 cells arising in acute infections expressed T-bet at distinct intensities and maintained their T-bet expression differences after viral clearance as memory cells for weeks in the steady state. However, it was unclear whether differential T-bet expression was associated with heterogeneity inside the Th1 population and if the quantitative T-bet memory, particularly of those cells expressing T-bet at low levels, could withhold the strong and continuous stimulation present during chronic infection.\n                <\/jats:p>\n                <jats:p>\n                  Using T-bet-ZsGreen reporter mice, virus-specific Th1 cells were characterized phenotypically at protein, RNA, and DNA\/chromatin accessibility levels. The Th1 cells arising during acute LCMV Armstrong infection showed a continuous spectrum of T-bet expression, ranging from cells with very high T-bet to cells with low T-bet. Even though the cells with low T-bet expression clearly possessed Th1 characteristics, they additionally showed certain Tfh-like features at protein and RNA level. When virus-specific Th1 cells were sorted according to T-bet-ZsGreen reporter expression intensity, adoptively transferred, and rechallenged by infecting the host animals with the chronic Clone 13 strain of LCMV, they maintained quantitative differences in T-bet reporter and IFN-\u03b3 expression levels. A subpopulation of the progeny of the former T-bet\n                  <jats:sup>low<\/jats:sup>\n                  cells still showed a mild Tfh-associated phenotype. Independent of their past and present T-bet expression level, all virus-reactive CD4\n                  <jats:sup>+<\/jats:sup>\n                  T cells acquired phenotypic signs of exhaustion as characterized by upregulation of PD-1, LAG3, and TOX and vast absence of effector cytokine co-expression in the chronic infection environment.\n                <\/jats:p>\n                <jats:p>\n                  Collectively, our findings highlight the heterogeneity of T-bet\n                  <jats:sup>+<\/jats:sup>\n                  antiviral CD4\n                  <jats:sup>+<\/jats:sup>\n                  T cells and the stability of quantitative differences in individual virus-specific CD4\n                  <jats:sup>+<\/jats:sup>\n                  T cells during chronic viral challenge infection.\n                <\/jats:p>","DOI":"10.1101\/2025.09.15.676263","type":"posted-content","created":{"date-parts":[[2025,9,17]],"date-time":"2025-09-17T01:40:18Z","timestamp":1758073218000},"source":"Crossref","is-referenced-by-count":0,"title":["Stable characteristics of intrapopulation heterogeneity in virus-specific Th1 cells during chronic viral challenge infection"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3987-9078","authenticated-orcid":false,"given":"Valerie","family":"Plajer","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-7924-6920","authenticated-orcid":false,"given":"Adri\u00e1n","family":"Madrigal-Avil\u00e9s","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2389-3222","authenticated-orcid":false,"given":"Maria","family":"Dzamukova","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4253-0387","authenticated-orcid":false,"given":"Nayar","family":"Dur\u00e1n-Hern\u00e1ndez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4916-7376","authenticated-orcid":false,"given":"Philippe","family":"Saikali","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vivien","family":"Holecska","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Isabel","family":"Panse","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katrin","family":"Lehmann","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jinfang","family":"Zhu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8015-6907","authenticated-orcid":false,"given":"Mir-Farzin","family":"Mashreghi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2946-8251","authenticated-orcid":false,"given":"Ahmed N.","family":"Hegazy","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6267-3452","authenticated-orcid":false,"given":"Caroline","family":"Peine","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6382-7281","authenticated-orcid":false,"given":"Max","family":"L\u00f6hning","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"issue":"1","key":"2025091914450791000_2025.09.15.676263v1.1","doi-asserted-by":"crossref","first-page":"108","DOI":"10.1016\/j.immuni.2014.12.018","article-title":"Individual T Helper Cells Have a Quantitative Cytokine Memory","volume":"42","year":"2015","journal-title":"Immunity"},{"key":"2025091914450791000_2025.09.15.676263v1.2","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2011.03.023"},{"key":"2025091914450791000_2025.09.15.676263v1.3","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2019.04.011"},{"key":"2025091914450791000_2025.09.15.676263v1.4","doi-asserted-by":"publisher","DOI":"10.1084\/jem.20180927"},{"key":"2025091914450791000_2025.09.15.676263v1.5","doi-asserted-by":"crossref","unstructured":"Kipps TJ , Parham P , Punt J , Herzenberg LA . 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